One of the greatest gaps in our knowledge of human development is at the time of embryo implantation at the initiation of pregnancy, due to limitations on the experimental use of human embryos. We have recently developed a paradigm with human embryonic stem cell-derived embryoid bodies which promotes the initiation of trophoblast differentiation and differentiated function by 3-dimensional culture in extracellular matrix environments. Yet, limitations remain in the direct applicability of this model to implantation in vivo. We thus propose to adapt this paradigm and expand it to an in vitro implantation model with IVF-produced rhesus monkey embryos with 2 specific aims: Specific Aim 1. To define trophoblast differentiation with rhesus monkey embryos in 3-dimensional extracellular matrix environments. Specific Aim 2. To determine the effects of 3-dimensional effector cells and selected growth factors on trophoblast differentiation. Few experimental models exist for research specifically relevant to human embryonic development and implantation. The unique structural organization of the primate placenta among mammals makes it difficult to extrapolate results from non-primate species to human implantation. The proposed studies will establish a nonhuman primate system to model placental development and morphogenesis and gain new insights into the parameters critical for pregnancy success. RELEVANCE TO HUMAN HEALTH: Failure to initiate appropriate early placental function (embryo attachment, trophoblast invasion, and initiation of hormone secretion) is likely to contribute to embryo loss in early pregnancy. Additionally, inappropriate placental development and abnormal establishment of the maternal-fetal interface are thought to contribute to the pathogenesis of diseases of later pregnancy (e.g., preeclampsia, fetal growth restriction). Finally, the intrauterine environment and prenatal fetal growth are now widely recognized to have a profound impact on adult endocrine, cardiovascular, metabolic and reproductive physiology. The development of new models with which we can investigate the early formation of the human placenta will be a major step in improving maternal-fetal health.